The present invention relates to novel, rapid and sensitive processes for determining antibiotics containing a xcex2-lactam ring in a biological fluid, using a receptor which is sensitive to the antibiotics containing a xcex2-lactam ring of Bacillus licheniformis. The invention also relates to kits for carrying out these processes.
At the present time, antibiotics are very widely used, not only as therapeutic agents in the treatment of infectious diseases caused by bacteria, but also as agents for storing foods and as additives in animal feed to stimulate growth. The need is thus increasingly being felt to be able to detect the presence of antibiotics, even in very low concentrations, in complex biological fluids, such as milk, urine, blood, serum, saliva, meat extracts, fermentation liquids or in buffered aqueous media.
The case of milk production is an example of this, since it is well known to use antibiotics to treat certain infectious diseases of dairy cattle.
However, for obvious medical reasons, milk intended for human consumption must, in principle, be free of any trace of antibiotics. Moreover, penicillin concentrations of 0.005 I.U./ml or less can have harmful effects during the manufacture of milk-based products such as cheese, yoghurt, etc.
Several situations may be envisaged. In a first case, for example to detect the presence of antibiotics at the farm before transferring into a wagon, priority will be given to an extremely rapid (less than 5 minutes) and simple test. It may also be possible to envisage using such a rapid test when, for example, the antibiotic which has been used for the treatment is known and when, moreover, this test allows the detection of the antibiotic in question at the legal standard. In the second case, when the emphasis is not on speed, the importance is to detect most, if not all, of the antibiotics at the legal standards.
The reason for this is that the laws in certain countries impose quite specific quality standards. For example, the US authorities require that the concentrations in milk of the following six antibiotics do not exceed quite specific values: penicillin, 5 ppb; ampicillin, 10 ppb; amoxicillin, 10 ppb; cloxacillin, 10 ppb; cephapirin, 20 ppb, ceftiofur, 50 ppb. The European Union imposes quality standards as follows: penicillin, 4 ppb; amoxicillin, 4 ppb; ampicillin, 4 ppb; cloxacillin, 30 ppb; dicloxacillin, 30 ppb; oxacillin, 30 ppb; cephapirin, 10 ppb, ceftiofur, 100 ppb; cefquinone 20 ppb; nafcillin 30 ppb; cefazoline, 50 ppb.
It may thus be advantageous to have access to a test which would allow most of the antibiotics to be detected. Moreover, in the dairy industry, it may be considered that, in the absence of a test which has all the characteristics of speed, sensitivity and simplicity, a test which would allow the best combination of these three parameters, even if they are not totally covered, would be advantageous.
Various types of tests have already been proposed for the detection of antibiotics containing a xcex2-lactam ring in a biological fluid.
These tests generally make use of detection methods which employ a recognition agent (receptor or antibody), which recognizes specifically the antibiotic or an analogue of this antibiotic, and a labelling agent (radioelement, enzyme, fluorescent agent, etc.), these agents being referred to hereinafter as detection reagents. Depending on the elements chosen, use is made of the terms radioimmunoassay (RIA), radioreceptor assay (RRA), enzyme immunoassay (EIA), etc. In their general principle, these tests employ the minimum combination of the two abovementioned elements (detection reagents) which will make it possible to obtain a result whose value is an indication of the quantity of analyte present.
It should be noted that, depending on the detection method selected, the labelling agent can be coupled alternatively to the recognition agent or to the antibiotic or to an analogue substance of the antibiotic in terms of its recognition by the recognition agent. There are also processes in which the recognition agent or the antibiotic or the analogue substance of the antibiotic contains, intrinsically, the labelling agent (for example, a radiolabelled analyte).
For dairy products, the analyte detection tests which are most widely described relate to the detection of antibiotics.
U.S. Pat. No. 4,239,852 describes a microbiological process for the detection in milk of antibiotics having a xcex2-lactam ring. According to this process, the sample of milk is incubated firstly in the presence of cell parts of a microorganism which is highly sensitive to antibiotics, and especially Bacillus stearothermophilus, and secondly in the presence of an antibiotic which is labelled (xe2x80x9ctaggedxe2x80x9d) with a radioactive element or with an enzyme. The incubation is conducted under conditions which allow antibiotics, if present in the sample, and the labelled antibiotic to bind to the cell parts.
Following incubation, the cell parts are separated from the mixture and then washed. Subsequently, the quantity of labelled antibiotic bound to the cell parts is determined and is compared with a standard. The quantity of labelled antibiotic bound to the cell parts is inversely proportional to the concentration of antibiotic present in the milk sample analysed.
This process requires fairly delicate handling, especially at the stage of separating the cell parts from the mixture. In addition, in its most sensitive version, which allows the detection of Penicillin G up to 0.01 I.U./ml and even up to 0.001 I.U./ml in milk, this process uses an antibiotic labelled with a radioactive element (14C or 125I). In this case, the determination of the quantity of antibiotic present or otherwise in the milk necessitates the use of a special instrument such as a scintillation counter, for example. In addition, handling radioactive products even in very small quantities is not completely free of risk for the person conducting the analysis.
European Patent Application 593 112 describes another method permitting the detection of antibiotics in milk. This method uses a protein isolated from an antibiotic-sensitive microorganism, such as Bacillus stearothermophilus. This protein is additionally labelled with an enzyme such as a peroxidase.
The test proceeds as follows: a sample of milk is incubated in a tube in the presence of the labelled protein; after incubation, the milk is transferred to a second tube on whose walls a reference antibiotic has been immobilized; a second incubation is carried out, and then the contents of the tube are removed; the walls of this second tube are washed three times with a wash solution, which is itself removed, and then the residues present in the second tube are transferred to a piece of absorbent paper; a colouring substrate is then added to the second tube, which is incubated once again, and then a solution which retards the development of the colour is added; the coloration of the tube is compared with the coloration of an identical test carried out in parallel on a standard sample of antibiotic. The quantity of labelled protein immobilized on the support, and therefore the intensity of the coloration, is inversely proportional to the quantity of antibiotic present in the milk sample analysed.
According to Example 1 of this patent application, this test makes it possible to detect penicillin G down to concentrations of the order of 5 ppb and makes it possible to detect amoxicillin (5 ppb), ampicillin (10 ppb), cephapirin (5 ppb) and ceftiofur (5 ppb). This test does not allow the detection of penicillin, amoxicillin and ampicillin up to the levels imposed by European Regulations and, on the other hand, this test is rather complex; it does not entirely fulfil the criteria of sensitivity and of simplicity sought in the context of the present invention.
However, the test is very tiresome to carry out, especially by unskilled personnel. Indeed, this test comprises numerous steps, including steps of transferring liquid and residues from one vessel to another, and a number of rinsing steps. Given the number and the type of steps required in this test, obtaining a reliable result depends heavily on the experimental know-how of the operative.
In addition, interpreting the result requires two tests to be carried out in parallel, thereby multiplying and further complicating the operations.
Other types of enzymatic processes have also been disclosed, which make it possible to determine low concentrations of antibiotics in milk (J. M. Frere et al., Antimicrobial Agents and Chemotherapy, 18(4), 506-510 (1980), and patents EP 85 667 and EP 468 946), which are based on the use of a specific enzyme, namely soluble exocellular D-alanyl-D-alanine carboxypeptidase, which is produced by Actinomadura R39 (designated xe2x80x9cenzyme R39xe2x80x9d hereinafter). Enzyme R39 possesses a specific activity of hydrolysing the D-alanyl-D-alanine groups of various peptides and is also capable of hydrolysing certain thioesters.
In addition, enzyme R39 reacts with antibiotics having a xcex2-lactam ring to form very rapidly an equimolar enzyme-antibiotic complex which is inactive and substantially irreversible.
In the most recent version of this test (EP 468 946) a predetermined volume of a sample of the liquid to be examined is incubated with a predetermined quantity of enzyme R39 under conditions which allow the xcex2-lactam antibiotic which may be present in the sample to react with the enzyme to form an equimolar enzyme-antibiotic complex which is inactive and substantially irreversible.
Subsequently, a predetermined quantity of thioester-type substrate is incubated with the product obtained in the first stage under conditions which allow the substrate to be hydrolysed by the residual enzyme R39 which has not been complexed with the antibiotic in the course of the first incubation. The quantity of mercaptoalkanoic acid thus formed is then determined by calorimetric assay with the aid of a reagent capable of producing a coloration by reaction with the free SH group of the mercaptoalkanoic acid. The intensity of the coloration is compared with a standard established beforehand from samples containing known quantities of antibiotics. Quantitative determination can be carried out by measurement in a spectrophotometer; in the case of milk, it may be necessary to clarify the sample beforehand.
According to the embodiments of patent EP 468,946, this process makes it possible to determine, in milk, 10 ppb of penicillin G for a total incubation time of 5 minutes and about 2.5 ppb of penicillin G for a total incubation time of 15 minutes.
Given the criteria of speed, simplicity and sensitivity required for the methods for detecting antibiotics in food products, the Applicant set itself the aim of investigating novel, even more effective methods for detecting antibiotics in a biological fluid. In particular, the Applicant set itself the aim of investigating methods for detecting, in a single test, most of the antibiotics whose content is regulated by the European and US authorities. Furthermore, the methods investigated should make it possible to obtain this result in a limited number of steps, which can preferably be carried out by unqualified personnel. The Applicant also investigated methods for achieving these aims with an incubation time which is shorter than that in the existing processes.
The Applicant has just discovered novel processes for detecting antibiotics containing a xcex2-lactam ring in a biological fluid, which allow these objectives to be achieved in a noteworthy manner.
Accordingly, the present invention relates to novel processes for detecting antibiotics containing a xcex2-lactam ring in a biological fluid, comprising the following steps
a) placing a determined volume of the said biological fluid in contact with an amount of recognition agent and incubating the mixture thus obtained under conditions which allow the complexation of the antibiotics, which may be present in the said biological fluid, with the recognition agent,
b) placing the mixture obtained in step a) in contact with at least one reference antibiotic immobilized on a support, under conditions which allow the complexation of the reference antibiotic with the amount of recognition agent which has not reacted in step a), and
c) determining the amount of recognition agent bound to the support, characterized in that the recognition agent comprises a receptor which is sensitive to the antibiotics containing a xcex2-lactam ring obtained from Bacillus licheniformis.